Pseudomonas putida

About: Pseudomonas putida is a research topic. Over the lifetime, 6854 publications have been published within this topic receiving 230572 citations.

Genetically Modified Bacterial Strains and Novel Bacterial Artificial Chromosome Shuttle Vectors for Constructing Environmental Libraries and Detecting Heterologous Natural Products in Multiple Expression Hosts

Abstract: The enormous diversity of uncultured microorganisms in soil and other environments provides a potentially rich source of novel natural products, which is critically important for drug discovery efforts. Our investigators reported previously on the creation and screening of an Escherichia coli library containing soil DNA cloned and expressed in a bacterial artificial chromosome (BAC) vector. In that initial study, our group identified novel enzyme activities and a family of antibacterial small molecules encoded by soil DNA cloned and expressed in E. coli. To continue our pilot study of the utility and feasibility of this approach to natural product drug discovery, we have expanded our technology to include Streptomyces lividans and Pseudomonas putida as additional hosts with different expression capabilities, and herein we describe the tools we developed for transferring environmental libraries into all three expression hosts and screening for novel activities. These tools include derivatives of S. lividans that contain complete and unmarked deletions of the act and red endogenous pigment gene clusters, a derivative of P. putida that can accept environmental DNA vectors and integrate the heterologous DNA into the chromosome, and new BAC shuttle vectors for transferring large fragments of environmental DNA from E. coli to both S. lividans and P. putida by high-throughput conjugation. Finally, we used these tools to confirm that the three hosts have different expression capabilities for some known gene clusters.

Cloning and molecular analysis of the Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) biosynthesis genes in Pseudomonas sp. strain 61-3.

Abstract: Polyhydroxyalkanoates (PHAs) are accumulated in various bacteria as intracellular carbon and energy storage material under nutrient-limited conditions (3, 26, 30) These bacterial PHAs are expected to become attractive alternatives for petrochemically based plastics, since they are biodegradable thermoplastics More than 90 different constituent monomer units have been found (47) The PHA-producing bacteria can be broadly divided into two groups according to the number of carbon atoms in the monomeric units of the PHAs produced (44) One group of bacteria, including Ralstonia eutropha (formerly Alcaligenes eutrophus), produces short chain length PHAs with C3 to C5 monomer units, while the other group, including Pseudomonas oleovorans, produces medium chain length PHAs with C6 to C14 monomer units (3, 43) Although the majority of bacteria accumulate either short chain length PHA or medium chain length PHA, several bacteria have been found to synthesize polyesters containing both short and medium chain length 3-hydroxyalkanoic acids (3HA) The bacteria Rhodospirillum rubrum (4), Rhodocyclus gelatinosus (27), and Rhodococcus sp (13) produced terpolyesters consisting of 3HA units of C4, C5, and C6 from hexanoate Aeromonas caviae produced a random copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx) (6, 8, 38) Pseudomonas strain GP4BH1 produced PHA containing 3HB and 3-hydroxyoctanoate (3HO) from octanoate and PHA containing 3HB, 3HO, and 3-hydroxydecanoate (3HD) from gluconate (46) In this bacterium, a polymer blend was suggested to be synthesized rather than a copolymer A recombinant strain of P oleovorans expressing R eutropha poly(3HB) [P(3HB)] biosynthesis genes has been shown to synthesize a blend of a P(3HB) homopolymer and a copolymer of 3HHx and 3HO units when grown on octanoate (49) Both polyesters were stored as separated granules within the cells (32) In addition, Pseudomonas fluorescens and several other Pseudomonas strains were found to produce a poly(3HB-co-3HA) [P(3HB-co-3HA)] copolymer consisting of 3HA units of C4 to C12 from 3HB and 1,3-butanediol (25) Although Thiocapsa pfennigii accumulated only a P(3HB) homopolymer from various carbon sources, a recombinant P putida strain harboring the PHA synthesis genes of T pfennigii produced a P(3HB-co-3HHx-co-3HO) terpolymer from octanoate (28) We have reported that Pseudomonas sp strain 61-3 isolated from soil produces a blend of a P(3HB) homopolymer and a random copolymer [P(3HB-co-3HA)] consisting of 3HA units of C4 to C12 from sugars and alkanoic acids (1, 19, 20) In addition, two different types of polyester granules were formed in the same cell (9, 21) This suggests that Pseudomonas sp strain 61-3 possesses two types of polyester synthases with different substrate specificities, that is, polyhydroxybutyrate (PHB) synthase and PHA synthase, specific for 3HB and 3HA units ranging from C4 to C12, respectively In this study, we cloned and sequenced the P(3HB) biosynthesis genes, as well as the P(3HB-co-3HA) biosynthesis genes, of Pseudomonas sp strain 61-3 The substrate specificity of each polyester synthase was evaluated by heterologous expression in PHA-negative mutants of P putida and R eutropha In addition, we found that the phbRPs gene product exhibits significant similarity to the AraC/XylS family of transcriptional activators and report that it is a positive regulatory protein that controls the expression of the P(3HB) biosynthesis operon

A genome-scale metabolic reconstruction of Pseudomonas putida KT2440: iJN746 as a cell factory.

Abstract: Background Pseudomonas putida is the best studied pollutant degradative bacteria and is harnessed by industrial biotechnology to synthesize fine chemicals. Since the publication of P. putida KT2440's genome, some in silico analyses of its metabolic and biotechnology capacities have been published. However, global understanding of the capabilities of P. putida KT2440 requires the construction of a metabolic model that enables the integration of classical experimental data along with genomic and high-throughput data. The constraint-based reconstruction and analysis (COBRA) approach has been successfully used to build and analyze in silico genome-scale metabolic reconstructions.

Gene Cloning and Characterization of Multiple Alkane Hydroxylase Systems in Rhodococcus Strains Q15 and NRRL B-16531

Abstract: The alkane hydroxylase systems of two Rhodococcus strains (NRRL B-16531 and Q15, isolated from different geographical locations) were characterized. Both organisms contained at least four alkane monooxygenase gene homologs (alkB1, alkB2, alkB3, and alkB4). In both strains, the alkB1 and alkB2 homologs were part of alk gene clusters, each encoding two rubredoxins (rubA1 and rubA2; rubA3 and rubA4), a putative TetR transcriptional regulatory protein (alkU1; alkU2), and, in the alkB1 cluster, a rubredoxin reductase (rubB). The alkB3 and alkB4 homologs were found as separate genes which were not part of alk gene clusters. Functional heterologous expression of some of the rhodococcal alk genes (alkB2, rubA2, and rubA4 [NRRL B-16531]; alkB2 and rubB [Q15]) was achieved in Escherichia coli and Pseudomonas expression systems. Pseudomonas recombinants containing rhodococcal alkB2 were able to mineralize and grow on C12 to C16 n-alkanes. All rhodococcal alkane monooxygenases possessed the highly conserved eight-histidine motif, including two apparent alkane monooxygenase signature motifs (LQRH[S/A]DHH and NYXEHYG[L/M]), and the six hydrophobic membrane-spanning regions found in all alkane monooxygenases related to the Pseudomonas putida GPo1 alkane monooxygenase. The presence of multiple alkane hydroxylases in the two rhodococcal strains is reminiscent of other multiple-degradative-enzyme systems reported in Rhodococcus.

Characterization of a phosphate solubilizing and antagonistic strain of Pseudomonas putida (B0) isolated from a sub-alpine location in the Indian Central Himalaya.

Abstract: The morphological, biochemical, and physiological characteristics of a phosphate solubilizing and antagonistic bacterial strain, designated as B0, isolated from a sub-alpine Himalayan forest site have been described. The isolate is gram negative, rod shaped, 0.8 × 1.6 μm in size, and psychrotrophic in nature that could grow from 0 to 35°C (optimum temp. 25°C). It exhibited tolerance to a wide pH range (3–12; optimum 8.0) and salt concentration up to 4% (w/v). Although it was sensitive to kanamycin, gentamicin, and streptomycin ( 1000 μg mL−1). The isolate showed maximum similarity with Pseudomonas putida based on 16S rRNA analysis. It solubilized tricalcium phosphate under in vitro conditions. The phosphate solubilization was estimated along a temperature range (4–28°C), and maximum activity (247 μg mL−1) was recorded at 21°C after 15 days of incubation. The phosphate solubilizing activity coincided with a concomitant decrease in pH of the medium. The isolate also exhibited antifungal activity against phytopathogenic fungi in Petri dish assays and produced chitinase, s-l,3-glucanase, salicylic acid, siderophore, and hydrogen cyanide. The plant growth promotion and antifungal properties were demonstrated through a maize-based bioassay under greenhouse conditions. Although the bacterial inoculation was found to result in significant increment in plant biomass, it stimulated bacterial and suppressed fungal counts in the rhizosphere. The present study is important with respect to enumerating microbial diversity of the colder regions as well as understanding the potential biotechnological applications of native microbes.